The hormonal form of vitamin D, calcitriol, acts through thevitamin D receptor (VDR) to regulate calcium homeostasis, cellproliferation, differentiation, and various immunefunctions. Defects in the target actions of calcitriol thus have a broadspectrum of manifestations ranging from hyperreactivity to a lack of effects. Hereditary resistance to calcitriol (HVDRR) usuallyresults from a mutation in the VDR gene and manifests as rickets.A collaborative study with Constantine Stratakis, NICHD,investigating the mutations in a family with HVDRR lead to theisolation of a bacterial artificial chromosome containing the VDRgene, and physical mapping of the VDR gene by fluorescent in situhybridization and radiation hybrid analysis to chromosome12cen-q12, flanked by markers SHGC 30216 and SHGC 9798.Furthermore, we used skin fibroblasts from subjects with HVDRR toexplore mechanisms of calcitriol action. These mutant cells display abnormalities in many discrete steps of the receptoractivation pathway. We used these natural knockout cells tostudy the functional domains of VDR involved in the control ofreceptor localization. New technologies were developed to explorethe mechanisms of hormone uptake and VDR movement. We cloned andstably expressed fully functional GFP cimeras of VDR and RXR inkidney and osteoblast cells. We also developed photostable, biologically active red fluorescing derivatives of vitamin D.Confocal laser scanning microscopy was used for real-timesimultaneous recordings of calcitriol and VDR import into thenucleus. These studies showed that hormone binds VDR both inthe cytoplasm and in the nucleus, and indicated that calcitriolinduces translocation of cytoplasmic VDR into thenucleus, and suggested that translocating VDR carry the hormoneinto the nucleus. Binding of the translocating VDR tomicrotubules were demonstrated in vivo, with microscopy in livingcells, and in vitro, by copolymerization andcoimmunoprecipitation experiments. We identified a new targetsite for VDR, the centrosome. Our studies with cytoskeletoninhibitors indicated that microtubule minus-end movement may playa role in the hormone-induced centrosomal targeting of VDR, aswell as the transcriptional activity of VDR. Using site-direcetedmutagenesis, we found that the integrity of the activationfunction 2 domain of VDR is essential for the hormone- inducedtranslocation of VDR into the nucleus and into the centrosome.Coexpression experiments with wilde-type and heterodimerizationmutant RXR revealed a role for heterodimerizing RXR in thehormone-independent translocationof VDR. Another collaborative study with Dr. G.D.Roodmans laboratory (Univ Texas Health Sci Center, San Antonio,TX) explored the mechanisms causing calcitriolhypersensitivity of osteoclast precursors from patients withPagets disease. Binding studies with our green fluorescenttagged calcitriol revealed an increased VDR affinity forcalcitriol in osteoclasts from Pagets patients, which may beresponsible for the enhanced calcitriol sensitivity in Pagetsdisease. We will continue to use GFP chimeras of wilde-type andmutant VDR, RXR and coactivators to gain further understanding ofthe disorders of the vitamin D endocrine system. - vitamin D receptor, vitamin D resistance rickets, calcitriol, confocal microscopy, microtubules, translocation, VDR gene, GFP, analogs